Department of Chemistry, Mount Holyoke College , South Hadley, Massachusetts 01075, United States.

Abstract

The cis-syn thymine cyclobutane dimer is a DNA photoproduct implicated in skin cancer. We compared the stability of individual base pairs in thymine dimer-containing duplexes to undamaged parent 10-mer duplexes. UV melting thermodynamic measurements, CD spectroscopy, and 2D NOESY NMR spectroscopy confirm that the thymine dimer lesion is locally and moderately destabilizing within an overall B-form duplex conformation. We measured the rates of exchange of individual imino protons by NMR using magnetization transfer from water and determined the equilibrium constant for the opening of each base pair K(op). In the normal duplex K(op) decreases from the frayed ends of the duplex toward the center, such that the central TA pair is the most stable with a K(op) of 8 × 10⁻⁷. In contrast, base pair opening at the 5'T of the thymine dimer is facile. The 5'T of the dimer has the largest equilibrium constant (K(op) = 3 × 10⁻⁴) in its duplex, considerably larger than even the frayed penultimate base pairs. Notably, base pairing by the 3'T of the dimer is much more stable than by the 5'T, indicating that the predominant opening mechanism for the thymine dimer lesion is not likely to be flipping out into solution as a single unit. The dimer asymmetrically affects the stability of the duplex in its vicinity, destabilizing base pairing on its 5' side more than on the 3' side. The striking differences in base pair opening between parent and dimer duplexes occur independently of the duplex-single strand melting transitions.

A. van’t Hoff analysis of changes in melting temperature (Tm) as a function of duplex concentration for parent (blue diamonds, bottom line) and dimer (red circles, top line) duplexes. Changes in the UV absorption at 260 nm were measured in 10X NMR buffer, and the Tm was determined to be the inflection point in the melting curves. Six or more samples were measured for each concentration, and data were fit to to determine the changes in enthalpy and entropy for duplex formation. B. Thermodynamic parameters for duplex formation for parent (blue) and dimer (red) duplexes. A gain in enthalpic stability with dimer formation is counterbalanced by a gain in entropic instability, leading to an overall change in free energy ΔΔG of +1.5 kcal/mol. C. CD spectra of single-strand 3(cyan), dimer-containing strand 3TT (orange), parent duplex 3–4 (blue), and dimer duplex 3TT-4 (red), plotted as ellipticity in degrees/cm*M base pairs.

A. 1D 1H spectrum of the parent duplex 3–4, measured in Exchange Buffer at 40 mM total ammonia species. Assignments are based on the 2D NOESY spectrum immediately below. B. Imino-imino region of the 2D NOESY spectrum for parent duplex 3–4, measured in 1X NMR buffer. The NOE walk though the crosspeaks shows the connections between stacked, adjacent thymine and guanine imino protons in the core of the duplex. The chain can be traced from G2 to G9; the terminal guanines G11 and G20 are not seen here. C. 1D 1H spectrum of the dimer duplex 3TT-4, measured in Exchange Buffer at 40 mM total ammonia species. D. Imino-imino region of the 2D NOESY spectrum for dimer duplex 3TT-4, measured in 1X NMR buffer. Note that the imino proton resonance for the 5’ thymine of the thymine dimer, T5, shifts markedly upfield from its position in the parent duplex; that of the 3’ thymine T6 moves more modestly.

The model of parent duplex 3–4 is shown from the minor and major grooves. Each diagram is color-coded as indicated in the legend to illustrate the magnitude in the differences in proton chemical shift between parent and dimer duplex. Wireframe structures in the default grey color indicate that the protons were not assigned (i.e. the H4’ and H5’ protons), and pale yellow wireframes indicate that the magnitude of the chemical shift change was less than ±.05 ppm. Protons represented by colored spheres experience a chemical shift change greater than ±.05 ppm. Arrows indicate the position of the thymine dimer lesion in the dimer duplex. Annotated NOESY spectra and tabulated values of the chemical shifts are given in the .

Imino Proton Resonances in the 1H NMR Spectra as a Function of Temperature

A. For the Parent Duplex 3–4, all of the imino peaks are visible to 28°C in 1X NMR buffer, though some change in chemical shift and line broadening does occur as expected. B. For Dimer Duplex 3TT-4, the duplex melts cooperatively between 23° and 28°C in 1X NMR buffer. Both dimer imino protons, T5 and T6, are visible below this range. Note that the ammonia-catalyzed imino proton exchange experiments are performed at 8°C.

The water protons are inverted via a 90° pulse, and the 1D spectrum of the imino protons is obtained after a range of 24 delay times between 520 µs and 2.5 s. Parent (A) and dimer (B) duplex spectra are shown here at five representative delay times at 2.9 mM ammonia base catalyst. These spectra illustrate how the resonances shrink or invert, then slowly return to equilibrium as the water and imino protons relax. At higher catalyst concentrations, proton exchange is more efficient and thus inversion of the resonances is more complete. The exchange rate of the imino protons (kex) is determined from plots of the amplitude of each peak as a function of time, as fit to .

For both the parent (A) and dimer (B) duplexes, the rate of exchange increases as the concentration of ammonia catalyst increases for all of the imino protons. Each imino proton is coded in matching colors and shapes between panels for ease of comparison. A fit of these data to gives the base pair opening equilibrium constant Kop.

Kop is shown for each internal base pair in the parent duplex 3–4 in the order in which each appears in the sequence. Two independent sample sets are shown for each duplex, measured at the concentrations given in the methods section. Kop values shown here are based upon a pKa for the thymine imino proton of 10.7; higher pKa values for the non-aromatic thymine dimer residues would lead to even larger values of Kop for T5 and/or T6.